ApoI NEB#R0566

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	AluI digestion was incubated at 37°C for 4 hours whereas ApoI was incubated at 50°C for overnight. In both digestions, heat inactivation for enzymes was given at 80°C/20 minutes. The restriction products were visualized on a 2.5 % agarose gel containing ethidium bromide. A consistent current at 0.75 m for 2.5 hrs were used for all agarose gel electrophoresis experiments to achieve consistency in RFLP fragment sizes.

Protocol tips

AluI digestion was incubated at 37°C for 4 hours whereas ApoI was incubated at 50°C for overnight. In both digestions, heat inactivation for enzymes was given at 80°C/20 minutes. The restriction products were visualized on a 2.5 % agarose gel containing ethidium bromide. A consistent current at 0.75 m for 2.5 hrs were used for all agarose gel electrophoresis experiments to achieve consistency in RFLP fragment sizes.

Publication protocol

To determine the level of pvrbp-2 polymorphism, RFLP analysis was carried out using two restriction enzymes ApoI and AluI (NEB Inc, USA). These enzymes were selected on the basis of maximum probability of enzymes cutting sites in the polymorphic region of pvrbp-2 and the feasibility to resolve digested PCR fragment on agarose gel. Virtual restriction mapping of pvrbp-2 was done using SeqBuilder module of DNA Lasergene 7.1 software for identification of suitable restriction enzymes for RFLP study. Four microliters of PCR product was digested with individual restriction enzyme. AluI digestion was incubated at 37°C for 4 hours whereas ApoI was incubated at 50°C for overnight. In both digestions, heat inactivation for enzymes was given at 80°C/20 minutes. The restriction products were visualized on a 2.5 % agarose gel containing ethidium bromide. A consistent current at 0.75 m for 2.5 hrs were used for all agarose gel electrophoresis experiments to achieve consistency in RFLP fragment sizes

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